During the production of recombinant proteins in mammalian cells, several modifications of the proteins take place after their expression (i.e., post-translationally) and, thus, result in a heterogeneity of the recombinant protein product. Since post-translational modifications are not encoded by the nucleotide sequence coding for the protein, but depend mostly on the action of several environmental enzymes, they are difficult to control and keep in tight (desired) specification ranges during the production. The biggest challenge during the manufacturing of especially recombinant therapeutic proteins is therefore to keep the features and quality of the protein product constant.
Within the comprehensive list of all relevant possible quality attributes of polypeptides, the C-terminal α-amidation is ascribed to the group of C-terminal heterogeneity. The processing of C-terminal lysine (Lys) on the heavy chain is one of the most common modifications of recombinant monoclonal antibodies (mAb). The Lys on the C-terminus of the heavy chain can be completely or partially removed by the activity of basic carboxypeptidases (CPs). Removal of one C-terminal Lys residue decreases the molecular weight by 128 Da and increases the positive charge by 1 unit. Partial removal of the C-terminal Lys residues results (due to the quaternary structure of mAbs) in a mixed population of antibodies with zero, one, or two C-terminal Lys residues (Liu et al. 2008). In the absence of Lys, the second amino acid glycine (Gly) can be further removed, and the remaining C-terminal amino acid proline (Pro) is subsequently enzymatically α-amidated.
C-terminal α-amidation is well known and has a major importance in the biologic activity of peptides and proteins. Many neuropeptides need a C-terminal α-amidation step to mature to the active, receptorbinding molecule. The non-amidated analog of leurotoxin for example has a fourfold lower activity in comparison to the leurotoxin comprising C-terminal α-amidated Pro. The stability of the human immunogenic MART-1 peptide in human plasma is markedly increased by C-terminal α-amidation. The C-terminal α-amidation of snake venom increases its lifetime in solution by decreasing its proteolytic degradation and leads to a higher activity in comparison to the unmodified peptide. The tetrapeptide Acetyl-Ser-Asp-Lys-Pro inhibits the proliferation of human and murine primitive hematopoietic cells and is easily degraded by Angiotensin I-Converting Enzyme (ACE). Its C-terminal α-amidated analog cannot be degraded by ACE and its plasma level is 26-fold higher than that of the wild type peptide. C-terminal α-amidation of peptides therefore increases their intrinsic biological and immunological activity, and also their stability.
C-terminal α-amidation is an exclusively enzymatic reaction which is catalyzed by the bifunctional peptidylglycine α-amidating (PAM) enzyme. The first step is the α-hydroxylation of the C-terminal glycine residue by the peptidylglycine α-hydroxylating monooxygenase (PHM, EC 1.14.14.3) domain. The second step is the dealkylation of the α-hydroxyglycine extended peptide to the α-amidated peptide and glyoxylate by the peptidyl α-hydroxyglycine α-amidatinglyase (PAL, EC 4.3.2.5) domain (Metha N M, 2009). For further reading of the enzymatic reaction of the PAM see the international applications PCT/EP2011/069756 or PCT/EP2013/057866, the disclosure content of which is incorporated herein by reference in its entirety.
In this context, several studies on the purified PAM enzyme were performed in order to increase the in vitro and in vivo C-terminal α-amidation. From those studies, a lot of different activators, inhibitors and cofactors were revealed.
EP 0409294A1 describes a substance which acts as a co-factor for the PAM enzyme, has no absorption spectrum above 225 nm, is not a peptide, is not ninhydrin-positive, has a molecular weight less than 1,000 Da, migrates to the positive pole upon electrodialysis and has an amphiphilic structure. The aforementioned inhibitor was used for the preparation of C-terminal α-amidated peptides or proteins (Gunther K, 1990).
Another cofactor for C-terminal α-amidation is described in GB 2233978A. The cofactor for the PAM enzyme is the compound having the formula R—(C═O)n—(CHOR1)n—CH2OR2 wherein R represents a hydrogen atom or an alkyl group, R1 and R2 represents a PO3H2 or SO3H group respectively (Gozzini et al., 1991).
In the paper of Bradbury and coworkers, the in vivo use of the PAM inhibitor 4-phenyl-3-butenoic acid was suggested. The availability of an in vivo inhibitor may allow controlling the degree of C-terminal α-amidation of certain peptide hormones, leading to the reduction in the circulating levels of the active form. In addition, the ability to control the C-terminal α-amidation in vivo may facilitate the studies of intracellular location, transport and storage of the α-amidating enzyme (Bradbury et al., 1989).
The hints that C-terminal heterogeneity can also influence certain properties of recombinant polypeptides are gathered from literature data on C-terminalα-amidated peptides. The process of C-terminal α-amidation elicited scientific and commercial attention by the recognition of the therapeutic potential of α-amidated peptides, such as calcitonin, oxytocine and vasopressin. In recent years, several efficient α-amidating reactions were developed, using preferentially the amidation reaction of pro-hormones catalyzed by an α-amidating enzyme (Kim K H and Seong B L, 2001). In this context, a bifunctional peptidylglycine α-amidating enzyme was successfully used for an in vitro reaction to convert C-terminal extended peptides into peptide hormones displaying a C-terminal α-amidated residue (Merkler D J, 1994). A process for the enzymatic conversion of C-terminal glycine extended peptides into the corresponding des-glycine peptide amide is described in GB 2 220 938 A (Castigliore et al., 1990).
However, little is still known of the effect of C-terminal α-amidation in larger polypeptides like antibodies. Even less or no knowledge is available about the influence of C-terminal heterogeneity of the recombinant polypeptides, such as glycoproteins, in terms of toxicology and immunogenicity, but some extrapolation from the above findings can be performed. Intrinsic antigenicity of the antibody may be altered if the modification changes the surface or the charge of the antibody protein. For antibody target peptides, Maillère and coworkers showed that C-terminal α-amidation of the target peptides provokes a dramatic effect on the corresponding antibody specificity. The antibody response in mice was drastically increased when the peptides showed a C-terminal α-amidation. From those data, the inventor of the present invention assumed that also therapeutic polypeptides, in particular therapeutic antibodies, with C-terminal α-amidation can have altered sensitizing (immunogenic) potential. From the evidence that C-terminal α-amidation increases the lifespan of the peptides and consequently also the T-cell response (Maillère et al., 1995), the extension of the half-life of the antibody may also have a considerable impact on the desired pharmacological efficacy as well as on a potential immunological response to the monoclonal antibody itself.
Thus, as the skilled artisan will appreciate, for the manufacture of recombinantly expressed therapeutic polypeptides such as antibodies, it is particularly important to control post-translational C-terminal modifications of the polypeptides produced, in order to provide i) a constant product quality and a constantly high yield, and/or ii) to increase the efficiency of the production process, and/or iii) to increase and/or fine-tune the physiological activity of the produced polypeptides and the safety of the derived drug, and/or iv) to match the post-translational features of a produced polypeptide to those of a reference polypeptide.
In view to the above, there is a need for a stable and controllable process of controlling or modulating C-terminal heterogeneity during the production of recombinant (glyco-) polypeptides by providing means for the regulation of the PAM and/or CP enzyme activity. Furthermore, there is also a need for high quality non-immunogenic recombinant polypeptides.
Thus, the problem underlying the present invention is the provision of means and methods to control the amount of C-terminal heterogeneity, in particular C-terminal α-amidation in (therapeutic) polypeptides.
The solution to said technical problem is achieved by providing the embodiments characterized in the independent claims of the present invention. The dependent claims are related to preferred embodiments. It is to be understood that value ranges delimited by numerical values are to be understood to include the said delimiting values.